The human and simian immunodeficiency viruses HIV, and SIV, are the causative agents of Acquired Immune Deficiency Syndrome (AIDS) and Simian Immunodeficiency Syndrome (SIDS), respectively. See Curren, J, et al., Science 329:1359-1357 (1985); Weiss, R. et al., Nature 324:572-575 (1986). The HIV virus contains an envelope glycoprotein, gp120 which binds to the CD4 protein present on the surface of helper T lymphocytes, macrophages and other cells. Dalgleish et al. Nature, 312:763 (1984). After the gp120 binds to CD4, virus entry is facilitated by an envelope-mediated fusion of the viral target cell membranes.
During the course of infection, the host organism develops antibodies against viral proteins, including the major envelope glycoproteins gp120 and gp41. Despite this humoral immunity, the disease progresses, resulting in a lethal immunosuppression characterized by multiple opportunistic infections, parasitemia, dementia and death. The failure of host anti-viral antibodies to arrest the progression of the disease represents one of the most vexing and alarming aspects of the infection, and augurs poorly for vaccination efforts based upon conventional approaches.
Two factors may play a role in the inefficiency of the humoral response to immunodeficiency viruses. First, like other RNA viruses (and like retroviruses in particular), the immunodeficiency viruses show a high mutation rate which allows antigenic variation to progress at a high rate in response to host immune surveillance. Second, the envelope glycoproteins themselves are heavily glycosylated molecules presenting few epitopes suitable for high affinity antibody binding. The poorly antigenic, "moving" target which the viral envelope presents, allows the host little opportunity for restricting viral infection by specific antibody production.
Cells infected by the HIV virus express the gp120 glycoprotein on their surface. Gp120 mediates fusion events among CD4.sup.+ cells via a reaction similar to that by which the virus enters the uninfected cell, leading to the formation of short-lived multinucleated giant cells. Syncytium formation is dependent on a direct interaction of the gp120 envelope glycoprotein with the CD4 protein. Dalgleish et al., supra, Klatzmann, D. et al., Nature 312:763 (1984); McDougal, J. S. et al. Science, 231:382 (1986); Sodroski, J. et al., Nature, 322:470 (1986); Lifson, J. D. et al., Nature, 323:725 (1986); Sodroski, J. et al., Nature, 321:412 (1986).
The CD4 protein consists of a 370 amino acid extracellular region containing four immunoglobulin-like domains, a membrane spanning domain, and a charged intracellular region of 40 amino acid residues. Maddon, P. et al., Cell 42:93 (1985); Clark, S. et al., Proc. Natl. Acad. Sci (USA) 84:1649 (1987).
Evidence that CD4-gp120 binding is responsible for viral infection of cells bearing the CD4 antigen includes the finding that a specific complex is formed between gp120 and CD4. McDougal et al., supra. Other workers have shown that cell lines, which were non-infective for HIV, were converted to infectable cell lines following transfection and expression of the human CD4 cDNA gene. Maddon et al., Cell 47:333-348 (1986).
In contrast to the majority of antibody-envelope interactions, the receptor-envelope interaction is characterized by a high affinity (K.sub.a =10.sup.8 l/mole) immutable association. Moreover, the affinity of the virus for CD4 is at least 3 orders of magnitude higher than the affinity of CD4 for its putative endogenous ligand, the MHC class II antigens. Indeed, to date, a specific physical association between monomeric CD4 and class II antigens has not been demonstrated.
In response to bacterial or other particle infection, the host organism usually produces serum antibodies that bind to specific proteins or carbohydrates on the bacterial or particle surface, coating the bacteria. This antibody coat on the bacterium or other particle stimulates cytolysis by Fc-receptor-bearing lymphoid cells by antibody-dependent cellular toxicity (ADCC). Other serum proteins, collectively called complement (C), bind to antibody-coated targets, and also can coat foreign particles nonspecifically. They cause cell death by lysis, or stimulate ingestion by binding to specific receptors on the macrophage called complement receptors. See Darnell J. et al., in Molecular Cell Biology, Scientific American Books, pp. 641 and 1087 (1986).
The most effective complement activating classes of human Ig are IgM and IgG1. The complement system consists of 14 proteins that, acting in order, cause lysis of cells. Nearly all of the C proteins exist in normal serum as inactive precursors. When activated, some become highly specific proteolytic enzymes whose substrate is the next protein in a sequential chain reaction.
The entire C sequence can be triggered by either of two initiation pathways. In one (the classic pathway), Ab-Ag complexes bind and activate C1, C4 and C2 to form a C3-splitting enzyme. In the second pathway, polysaccharides commonly on the surface of many bacteria and fungi bind with trace amounts of a C3 fragment and then with two other proteins (factor B and properdin) to form another C3-splitting enzyme. Once C3 is split by either pathway, the way is open for the remaining sequence of steps which lead to cell lysis. See Davis, B. D., et al., In Microbiology, 3rd ed., Harper and Row, Philadelphia, Pa., pp. 452-466 (1980).
A number of workers have disclosed methods for preparing hybrid proteins. For example, Murphy, U.S. Pat. No. 4,675,382 (1987), discloses the use of recombinant DNA techniques to make hybrid protein molecules by forming the desired fused gene coding for a hybrid protein of diptheria toxin and a polypeptide ligand such as a hormone, followed by expression of the fused gene.
Many workers have prepared monoclonal antibodies (Mabs) by recombinant DNA techniques. Monoclonal antibodies are highly specific well-characterized molecules in both primary and tertiary structure. They have been widely used for in vitro immunochemical characterization and quantiation of antigens. Genes for heavy and light chains have been introduced into appropriate host and expressed, followed by reaggregation of the individual chains into functional antibody molecules (see, for example, Munro, Nature 312:597 (1984); Morrison, S. L., Science 229:1202 (1985); Oi et al., Biotechniques 4:214 (1986); Wood et al., Nature 314:446-449 (1985)). Light- and heavy-chain variable regions have been cloned and expressed in foreign hosts wherein they maintained their binding ability (Moore et al., European Patent Application 0088994 (published Sep. 21, 1983)).
Chimeric or hybrid antibodies have also been prepared by recombinant DNA techniques. Oi and Morrison, Biotechniques 4:214 (1986) describe a strategy for producing such chimeric antibodies which include a chimeric human IgG anti-leu3 antibody.
Gascoigne, N. R. J., et al., Proc. Natl. Acad. Sci. (USA) 84:2936-2940 (1987) disclose the preparation of a chimeric gene construct containing a T-cell receptor .alpha.-chain variable (V) domain and the constant (C) region coding sequence of an immunoglobulin .sub..gamma. 2a molecule. Cells transfected with the chimeric gene synthesize a protein product that expresses immunoglobulin and T-cell receptor antigenic determinants as well as protein A binding sites. This protein associates with a normal .lambda. chain to form an apparently normal tetrameric (H.sub.2 L.sub.2, where H=heavy and L=light) immunoglobulin molecule that is secreted.
Sharon, J., et al., Nature 309:54 (1984), disclose construction of a chimeric gene encoding the variable (V) region of a mouse heavy chain specific for the hapten azophenylarsonate and the constant (C) region of a mouse kappa light chain (V.sub.H C.sub.K). This gene was introduced into a mouse myeloma cell line. The chimeric gene was expressed to give a protein which associated with light chains secreted from the myleoma cell line to give an antibody molecule specific for azophenylarsonate.
Morrison, Science 229:1202 (1985), discloses that variable light- or variable heavy-chain regions can be attached to a non-Ig sequence to create fusion proteins. This article states that the potential uses for the fusion proteins are three: (1) to attach antibody specifically to enzyme for use in assays; (2) to isolate non-Ig proteins by antigen columns; and (3) to specifically delivery toxic agents.
Recent techniques for the stable introduction of immunoglobulin genes into myeloma cells (Banerji, J., et al., Cell 33:729-740 (1983); Potter, H., et al., Proc. Natl. Acad. Sci. (USA) 81:7161-7165 (1984)), coupled with detailed structural information, have permitted the use of in vitro DNA methods, such as mutagenesis, to generate recombinant antibodies possessing novel properties.
PCT Application WO87/02671 discloses methods for producing genetically engineered antibodies of desired variable region specificity and constant region properties through gene cloning and expression of light and heavy chains. The mRNA from cloned hybridoma B cell lines which produce monoclonal antibodies of desired specificity is isolated for cDNA cloning. The generation of light and heavy chain coding sequences is accomplished by excising the cloned variable regions and ligating them to light or heavy chain module vectors, This gives cDNA sequences which code for immunoglobulin chains. The lack of introns allows these cDNA sequences to be expressed in prokaryotic hosts, such as bacteria, or in lower eukaryotic hosts, such as yeast.
The generation of chimeric antibodies in which the antigen-binding portion of the immunoglobulin is fused to other moieties has been demonstrated. Examples of non-immunoglobulin genes fused to antibodies include Staphylococcus aureus nuclease, the mouse oncogene c-myc, and the Klenow fragment of E. coli DNA polymerase I (Neuberger, M. S., et al., Nature 312:604-612 (1984); Neuberger, M. S., Trends in Biochemical Science, 347-349 (1985)). European Patent Application 120,694 discloses the genetic engineering of the variable and constant regions of an immunoglobulin molecule that is expressed in E. coli host cells. It is further disclosed that the immunoglobulin molecule may be synthesized by a host cell with another peptide moiety attached to one of the constant domains. Such peptide moieties are described as either cytotoxic or enzymatic. The application and the examples describe the use of a lambda-like chain derived from a monoclonal antibody which binds to 4-hydroxy-3-nitrophenyl (NP) haptens.
European Patent Application 125,023 relates to the use of recombinant DNA techniques to produce immunoglobulin molecules that are chimeric or otherwise modified. One of the uses described for these immunoglobulin molecules is for whole-body diagnosis and treatment by injection of the antibodies directed to specific target tissues. The presence of the disease can be determined by attaching a suitable label to the antibodies, or the diseased tissue can be attacked by carrying a suitable drug with the antibodies. The application describes antibodies engineered to aid the specific delivery of an agent as "altered antibodies."
PCT Application WO83/101533 describes chimeric antibodies wherein the variable region of an immunoglobulin molecule is linked to a portion of a second protein which may comprise the active portion of an enzyme.
Boulianne et al., Nature 312:643 (1984) constructed an immunoglobulin gene in which the DNA segments that encode mouse variable regions specific for the hapten trinitrophenol (TNP) are joined to segments that encode human mu and kappa regions. These chimeric genes were expressed to give functional TNP-binding chimeric IgM.
Morrison et al., P.N.A.S. (USA) 81:6851 (1984), disclose a chimeric molecule utilizing the heavy-chain variable region exons of an anti-phosphoryl choline myeloma protein G, which were joined to the exons of either human kappa light-chain gene. The genes were transfected into mouse myeloma cell lines, generating transformed cells that produced chimeric mouse-human IgG with antigen-binding function.
Despite the progress that has been achieved on determining the mechanism of HIV infection, a need continues to exist for methods of treating HIV viral infections.